The invention relates to solvents for biopolymer synthesis and the use of such solvents for assembling an array of biopolymers on a solid support.
Genetic information generated by the Human Genome Project is allowing scientists, physicians, and others to conduct diagnostic and experimental procedures on an unprecedented scale in terms of speed, efficiency, and number of screenings performed within one procedure. In order to make full use of this new information, there is an urgent need for the ability to screen a large number of chemical compounds, particularly oligonucleotide probes, against samples of DNA or RNA from normal or diseased cells and tissue. One important tool for such analyses is nucleic acid hybridization, which relies on the difference in interaction energies between complementary and mismatched nucleic acid strands (see U.S. Pat. No. 5,552,270 to Khrapko et al.). Using this tool, it is possible to determine whether two short pieces of nucleic acid are exactly complementary. Longer nucleic acids can also be compared for similarity.
Nucleic acid hybridization is often used for screening cloned libraries to identify similar, and thus presumably related, clones. This procedure typically involves using natural nucleic acid targets which are usually bound to a membrane, and a natural or synthetic nucleic acid probe which is washed over many targets at once. With the appropriate mechanics, membranes can be constructed with targets at a density of generally between one and ten targets per mm2. Hybridization detection is carried out by labeling the probe, for example either radioactively or with chemiluminescent reagents, and then recording the probe""s emissions onto film.
Alternative approaches to nucleic acid hybridization have involved oligonucleotide probes that are synthesized on a solid support or a substrate, and then hybridized to a single natural target. While such alternative approaches have the potential for large-scale assembly of oligonucleotide arrays, the cost of making such a variety of arrays is prohibitive.
Recently, there have been reports of using microdrop dispensers to generate oligomers and polymers arranged, on a substrate, in arrays of microdroplets:
For example, T. Brennan, Human Genome Program, U.S. Department of Energy, Contractor-Grantee Workshop III, Feb. 7-10, 1993, Santa Fe, N.M., Methods to Generate Large Arrays of Oligonucleotides 92 (1993), describes that arrays of oligonucleotides were sought to be synthesized in parallel chemical reactions on glass plates, using arrays of piezoelectric pumps, similar to an inkjet printer, as a means for delivering reagents. In such a scheme, each array element is separated by its neighbor by a perfluoroalkane tension barrier which is not wet by the acetonitrile reaction solvent.
U.S. Pat. No. 5,449,754 to Nishioka describes that peptide arrays can be obtained using an inkjet print head to deposit a dimethylformamide solution of N-protected activated amino acids, in the form of microdroplets, onto an aminosilylated glass slide which is subsequently washed with a trifluoroacetic acid solution to remove the N-protecting groups from the anchored amino acids. The process is repeated until amino acids having the desired sequence are obtained.
U.S. Pat. No. 5,474,796 to Brennan describes a piezoelectric impulse jet pump apparatus for synthesizing arrays of oligomers or polymers having subunits connected by ester or amide bonds. According to that scheme, a glass plate is coated with a fluoropolymer which is then selectively removed, leaving glass regions, in spots upon which oligomer or polymer synthesis would take place. The glass regions are epoxidized and subsequently hydrolyzed to afford a hydroxyalkyl group that would react with an activated chemical species. Where the oligomers sought to be synthesized are oligonucleotides, microdroplets of acetonitrile or diethyleneglycol dimethyl ether solutions of 5xe2x80x2-protected nucleotide monomers that are activated at their 3xe2x80x2-positions would be dispensed via a piezoelectric jet head, and would impinge upon the hydroxyalkyl group, forming a covalent bond therewith. After removing the 5xe2x80x2-protecting groups by flooding the surface of the plate with a deprotecting reagent, the process is repeated until the desired oligonucleotides are obtained.
International Publication No. WO 95/25116 by Baldeschwieler et al. describes a method for chemical synthesis at different sites on a substrate using an inkjet printing device to deliver reagents to specific sites of the substrate. The inkjet printing device is envisioned to deposit,.in repeatable sequence, (a) a protected molecule onto the substrate, (b) a deprotecting reagent onto the protected molecule so as to expose a reactive site, and (c) a second protected molecule at the site of the now-deprotected molecule, so as to form a growing chain of molecules. According to this publication by Baldeschwieler et al., useful reaction solvents are dibromomethane, nitromethane, acetonitrile and dimethylformamide.
U.S. Pat. No. 5,658,802 to Hayes et al. describes a dispensing apparatus that is allegedly capable of providing droplets having a volume of 10 pL to 100 pL, and purportedly useful for synthesizing arrays of diagnostic probes. According to that reference, the dispensing apparatus is capable of dispensing xe2x80x9cliquidsxe2x80x9d that may contain DNA molecules, peptides, antibodies, antigens, enzymes or entire cells; however, no specific examples of such xe2x80x9cliquidsxe2x80x9d are disclosed.
The dispensation of certain organic solvents from an inkjet printing device for use in chemical synthesis has several drawbacks. First, many organic solvents, such as alcohols or amines, bear functional groups that are capable of reacting with those chemical compounds sought to be dispensed from the inkjet device. Second, solvents having low boiling points are relatively volatile, and can evaporate from a substrate before the reactant(s) dissolved therein have completely reacted with any species bound to the substrate. Third, such volatile solvents can begin to evaporate at the site of the inkjet print head, causing reactants dissolved in the solvents to precipitate and clog the inkjet nozzle. Fourth, solvents that have low surface tension values have a relatively high affinity for the face of the inkjet nozzle, and tend to give rise to unstable and non-uniformly sized droplets. Fifth, solvents that have low viscosity values tend to form non-uniformly sized droplets due to their response to residual oscillations in the solvent. Sixth, many organic solvents, particularly acetonitrile, have the highly undesirable characteristic of being capable of dissolving adhesives and plastics used in inkjet print heads. Thus, prior to the present invention the organic solvents used for synthesizing oligonucleotides were ineffective in automated systems employing plastic components such as ink jet print heads.
The use of inkjet printing technology in chemical synthesis would be particularly useful for synthesis of a large number of different biopolymer species, such as oliognucleotides. Using a manual approach to synthesize each species would be prohibitively time consuming.
Thus, there exists a need for a method of synthesizing an array of oligonucleotides on a solid support that can be automated. In particular, there exists a need for a class of organic solvents, useful for chemical synthesis, that is relatively inert, and that has boiling point, surface tension and viscosity properties that are optimal for microdroplet formation from an inkjet device. The present invention satisfies such needs and provides related advantages as well.
Citation of any references above shall not be construed as an admission that such reference is available as prior art to the present application.
The invention provides a method of oligonucleotide synthesis. The method consists of chemically coupling a first nucleotide to a second nucleotide in a high surface tension solvent. Synthesis can be performed either through the coupling of the 5xe2x80x2 position of the first nucleotide to the 3xe2x80x2 position of the second nucleotide or vice versa. The high surface tension solvent can be, for example, propylene carbonate and can be used in a variety of nucleotide coupling reactions including, for example, phosphodiester, phosphotriester, phosphite triester or phosphoramidite and H-phosphonate chemistries. Additionally, the high surface tension solvent can be used with deoxyribonucleotide or ribonucleotide monomers as well as with modified nucleotides or nucleotide derivatives. The high surface tension solvent can be used with iterative coupling cycles to synthesize oligonucleotides of a desired length and sequence. Also provided is a method of oligonucleotide synthesis using a high surface tension solvent wherein the synthesis is automated.
The invention also provides a microdroplet of a solution, the solution comprising a solvent having a boiling point of 150xc2x0 C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above.
The invention further provides a method for dispensing microdroplets of a solution from a microdroplet dispensing device, the microdroplet dispensing device comprising (a) a manifold which contains the solution, (b) a nozzle at one end of the manifold and (c) means for applying a pressure pulse to the manifold, the means located at the other end of the manifold, comprising the step of applying a pressure pulse to the manifold, thereby dispensing the solution through the nozzle in microdroplet form, the solution comprising a high surface tension solvent.
The invention still further provides a method for chemical synthesis, comprising the step of dispensing a microdroplet of a solution comprising (i) a first chemical species and (ii) a solvent, such that the microdroplet impinges a second chemical species and forms a third chemical species therewith, the solvent having a high surface tension.
The invention also provides a fully automated solution for synthesizing oligonucleotides, particularly deoxyribonucleosides and ribonucleosides, by repeatedly cycling a substrate through steps of depositing nucleoside monomers and of treating the substrate by rinsing off unattached nucleoside monomers. A system in accordance with the invention includes an inkjet print head for spraying nucleoside monomers on a substrate, a scanning transport for moving the substrate with respect to the print head so that the monomer is deposited at specified sites, a flow cell for treating the substrate deposited with the monomer by exposing the substrate to selected fluids, a treating transport for moving the substrate between the print head and the flow cell for treatment in the flow cell, and an alignment unit for aligning the substrate so that the substrate is correctly positioned with respect to the print head each time the substrate is positioned for deposition. Computer-controlled motion stages and vacuum chucks are used to move the substrate during deposition and to move the substrate between the print head and the flow cell.
Each time the substrate is picked up by a vacuum chuck and placed over the print head, the substrate is positionally calibrated by using a camera in conjunction with marks that are placed on the substrate the first time it is handled. Translational misalignment is corrected by moving the vacuum chuck in two axes of linear motion. Rotational misalignment is corrected by physically rotating the vacuum chuck within a substrate holder.
Software, programmed apparatuses, and computer readable memory, for carrying out the methods of the invention are also provided.
The present invention may be understood more fully by reference to the following figures, detailed description and illustrative examples which are intended to exemplify non-limiting embodiments of the invention.